The efficiency of stable retroviral transduction is a critical step, which often determines the success or failure of the gene therapy approaches to treatment of human diseases. To successfully transduce a gene into host cells, retroviral vectors must integrate into the host cell genome. Retroviral DNA integration involves a staggered cut of host cell DNA followed by joining of S'-ends of viral DNA to host DNA by the retroviral enzyme integrase. The integration process thus creates short, single-strand DNA gaps that flank retroviral DNA. Complete, stable transduction of the viral DNA then depends on post-integration repair, which follows the integrase-mediated 3'-end joining reaction. The long-term goal of our laboratory is to understand the role of host cell proteins in this essential, but incompletely characterized repair step in the viral life cycle. This proposal builds on our discoveries that post-integration repair depends on the proteins of the non- homologous end joining (NHEJ) DNA repair pathway (Aim 1), and on the cellular histone deacetylase 4 (HDAC4) protein (Aim 2). Both of these discoveries will be put to use in Aim 3. The experiments described in this proposal should enhance our understanding of the roles of the proteins in post-integration repair, and pave the way to development of novel methods to increase success rate of gene therapy using retroviral vectors. In the Aim 1, we will further dissect the process of post-integration repair in NHEJ-deficient and normal cells and determine, if specific NHEJ proteins are required for specific steps of the post-integration repair, which include filling of DNA gaps, trimming of short viral DNA flaps, ligation of the newly exposed 5'- ends of viral DNA to host DNA and reconstitution of appropriate chromatin structure and composition. Results from the proposed experiments should not only deepen our understanding of how NHEJ proteins facilitate post-integration repair, but also significantly enhance our understanding of NHEJ function in important cellular pathways. In the Aim 2, we will pursue our new findings that stable transduction by retroviral vectors requires the cellular HDAC4 protein. In addition, we present evidence indicating that HDAC4 accumulates at sites of retroviral DNA integration. These novel findings reveal HDAC4 as a new player in the retroviral life cycle. Using methodologies developed for the Aim 1, in the Aim 2 we will test the hypothesis that HDAC4 is required for stable transduction through enhancement of post-integration repair. Analysis of the HDAC4 function in retroviral transduction should increase our understanding of the overall role of HDAC4 in DNA repair and again allow us a new avenue to augment the efficiency of retroviral vectors. In the final Aim of this proposal (Aim 3), we will test the hypothesis that the efficiency of stable retroviral transduction can be enhanced by increasing the intracellular amounts and activities of cellular proteins, which are required for efficient post-integration repair. The experiments described in this Aim should lead to development of new methods to increase efficiency and success rate of gene therapy using retroviral vectors.